Immunization with polyvalent venom vaccines

ABSTRACT

This disclosure relates to materials and methods useful for vaccinating mammals against the effects of envenomation by venomous organisms (including the Western Rattlesnake) by making use of venom from multiple distinct populations, subspecies or species of the organism, to make a vaccine more broadly protective against other populations, subspecies or species.

TECHNICAL FIELD

This invention relates to the use of toxoid snake venom or other toxoidvenoms for vaccinating mammals against the effects of envenomation bymultiple species of venomous snakes or other animals, by using toxoidsderived from snakes or animals sourced from multiple geographicallocations.

BACKGROUND

A wide variety of organisms (including certain species of snakes;spiders, scorpions and other arachnids; insects; fish; jellyfish; andlizards) are capable of producing venoms, and of transmitting them(“envenomation”) by either biting or stinging.

Rattlesnake venom is a complex mixture of toxic components (individuallyreferred to as toxins), composed mainly of both enzyme and non-enzymeproteins. Toxins are divided into more than twenty different proteinfamilies that are defined by their molecular structure. While familiesof toxins share a general structure, there may be a variety of toxicactivities represented by a single toxin family. Typically, anindividual rattlesnake's venom will contain toxins representing a dozentoxin families and can contain upwards of 50 unique toxins. Whilelethality is a major concern in envenomation, other serious adverseevents that are likely, include hemorrhage, tissue necrosis, andsystemic blood clotting, among others.

A vast majority of snakebite envenomation cases in the state ofCalifornia result from encounters with individuals from the twosubspecies of Western Rattlesnake, the Northern Pacific Rattlesnake(Crotalus oreganus oreganus) and the Southern Pacific Rattlesnake(Crotalus oreganus helleri). The venom of this species can cause a widerange of clinical issues, including tissue damage, blood clotting, andeffects on the nervous system.

One method of acutely treating domestic animals or humans that have beenenvenomated by venomous snakes or other venomous organisms is toadminister an intravenous dose of monoclonal or polyclonal antibodiesharvested from a mammalian species (e.g. sheep, horse, or goat) that hasbeen immunized against a venom. Antibody-based therapeutics are known asantivenoms or antivenins.

Snake antivenom is “monovalent” if it is produced from the venom of asingle species of snake and “polyvalent” if produced from the venom ofmultiple species of snakes.

While the use of antivenom is an acute treatment option used followingan envenomation event, vaccination against venom is a prophylactic—forprotecting an animal or human before they suffer envenomation. Alltoxins can be denatured by physical or chemical means to produceattenuated materials known as toxoids, which immunologically resemblethe source toxin and can produce a protective immune response. Whiletoxoids closely resemble the original toxins in their structure, theneutralization process(es) disrupt enough molecular structure to renderthe toxoids nontoxic.

The vaccination of a mammal against the effects of envenomation can beaccomplished using either bioactive venom toxins or venom toxoids.Toxoids are preferred to minimize the risk of injury or death to themammal from toxin. Toxoids are preferably administered at a dose andfrequency to generate a strong and lasting immune response. Thus indesigning a toxoid vaccine, the goal is to present the patient's immunesystem with enough toxoids to stimulate the immune system to defendagainst each of the potentially dangerous components (or itsimmunological equivalent) that might be transmitted in envenomation.

Snake venom is known to vary within and between species. There are morethan 30 distinct species of venomous snakes in the contiguous U.S., eachwith a distinct venom composition compared with other species and somewith distinct compositions between subspecies. Existing research alsoshows that snake venom varies within and between geographic populationsof venomous snake. This immense amount of variation is thought to arisefrom locality specific evolutionary pressures acting on snake venomcomposition and affecting the ecological fitness of a population overtime.

The Southern Pacific Rattlesnake (Crotalus oreganus helleri) rangesacross some of the most heavily populated areas of Southern California.A number of geographically separate populations of the Southern PacificRattlesnake exhibit significant variations in venom composition, eachwith unique biochemical properties. The Northern Pacific Rattlesnake(Crotalus oreganus oreganus) ranges further north, and shows localvariations in venom composition.

There is only one commercially-available snake venom vaccine (Canine andEquine Rattlesnake Toxoid Vaccine, from Red Rock Biologics, Woodland,Calif.) approved by the USDA-CVB for use in domesticated animals (dogsand horses). The vaccine is monovalent and is produced from a singlespecies—the Western Diamondback Rattlesnake (Crotalus atrox). Thisproduct is referred to as “CAT Vaccine” (Crotalus atrox toxoid vaccine).

The CAT vaccine is a sub-lethal dose of a mixture of toxoids that causesthe vaccinated mammal to mount an immune response and produce its ownanti-venom antibodies against future snakebite. Antibodies produced byan animal immunized with the CAT vaccine have limited or no ability toprotect against the venom of species of rattlesnake other than theWestern Diamondback Rattlesnake (Crotalus atrox). See Cates et al.(2015), Comparison of the protective effect of a commercially availableWestern Diamondback Rattlesnake toxoid vaccine for dogs againstenvenomation of mice with Western Diamondback Rattlesnake (Crotalusatrox), Northern Pacific Rattlesnake (Crotalus oreganus oreganus), andSouthern Pacific Rattlesnake (Crotalus oreganus helleri) venom, Am. J.Vet. Res. 76(3):272-9.

The failure of this CAT Vaccine to protect mice, an experimental modelmammal, when experimentally envenomated with the venom from a SouthernPacific Rattlesnake (Crotalus oreganus helleri) presumably results fromdifferences between the venom used to formulate the CAT vaccine and thevenom of the Southern Pacific Rattlesnake.

The derived heterodimeric lectin toxins (α- and β-chains) characteristicof viper venoms, which exhibit a diversity of biological activitiesincluding anticoagulation and agonism/antagonism of platelet activationor procoagulation, are both absent from the San Jacinto Mountainpopulation, but are present in all other populations of the SouthernPacific Rattlesnake. The extreme variation of venom composition betweenthe different populations of Southern Pacific Rattlesnake and theabsence of neurotoxin phospholipase A2 in the CAT vaccine renders theCAT vaccine ineffective against not only all of the Southern PacificRattlesnake populations, including the San Jacinto Mountain population,but also against envenomation from other species of rattlesnakeincluding the Mojave Rattlesnake (Crotalus scutulatus).

Thus, there is currently a great need for polyvalent and broadlyprotective venom toxoid vaccines in the regions where dangerous snakesreside. A broadly protective vaccine is expected to significantly reducemortality of dogs and horses from snakebite envenomation, significantlyreduce the suffering of canine and equine patients and their owners, andsignificantly reduce the financial burden to dog and horse owners byreducing the duration of treatment and hospitalization of envenomatedanimals.

SUMMARY

The preferred embodiment relates to: a polyvalent vaccine that includestoxoids derived from the venom of multiple rattlesnake species orsourced from rattlesnakes from distinct geographical regions, which willprotect a mammal from envenomation by such species in addition tosubspecies and other species. The desired vaccine will protect againstrattlesnakes indigenous to a particular geographical region, andpreferably, subspecies and other species of rattlesnakes, and morepreferably, all species of rattlesnakes and some other species ofvenomous snake.

The invention also relates to a polyvalent vaccine with toxoids derivedfrom the venom of a variety of snakes and scorpions native to the Asiaand Africa, including the Palestine Yellow Scorpion (Leiurusquinquestriatus), fat-tailed scorpions such as the Black Scorpion(Androctonus crassicauda) and A. amoreuxi, as well as Buthacusarenicola, Buthus occcitanus, and the Egyptian Cobra (Naja haje), theBlack Desert Cobra (Walterinnesia aegyptia), the Puff Adder (Bitisarietans), the Painted Saw-Scaled Viper (Echis coloratus), the IndianSaw-Scaled Viper (E. carinatus), and the Saharan Horned Viper (Cerastescerastes). These venoms can be denatured, mixed and prepared as abroadly-protective vaccine against envenomation by these organisms, andrelated species, using the technology, methodologies, methods, processesand practices described herein.

The invention also relates to a polyvalent viral vaccine formosquito-borne viruses including the West Nile virus, chikungunya virus,the four common variants of dengue virus, and the Zika virus. Theseviruses can be denatured and prepared as a broadly-protective vaccineagainst several mosquito-borne viruses, using the technology,methodologies, methods, processes and practices described herein.

A preferred embodiment also relates to venom combinations frompopulations of Western Rattlesnakes (Crotalus oreganus ssp.) so thatvenom combinations from these distinct populations will include alldangerous components (or their immunological equivalent) to which amammal could be exposed upon envenomation, and then using this combinedvenom to produce more effective toxoid rattlesnake vaccines for at-riskmammals in the relevant range of such species. The vaccine wouldpreferably be protective against all subspecies of Western Rattlesnake,including the Southern Pacific Rattlesnake and the Northern PacificRattlesnake, and will preferably also provide some degree of crossprotection against envenomation by any pit viper in the United States,including the Mojave Rattlesnake (Crotalus scutulatus), all subspeciesof the Prairie Rattlesnake (Crotalus viridis ssp.), Eastern DiamondbackRattlesnake (Crotalus adamanteus), Western Diamondback Rattlesnake(Crotalus atrox), Red Diamondback Rattlesnake (Crotalus ruber ssp.),Timber Rattlesnake (Crotalus horridus), Massasauga (Sistrurus ssp.),Sidewinder (Crotalus cerastes), water moccasins (Agkistrodon sp.), andcopperheads (Agkistrodon sp.).

The venom combinations can be treated to make vaccines by any of anumber of methods well-known to those skilled in the art, and thenstored for administration by: simply refrigerating or freezing a liquidvenom mixture, lyophilizing such liquid mixture, adding alum or one ormore other adjuvant materials to such liquid mixture and storing itrefrigerated or frozen or lyophilizing the mixture, or by any other suchwell-known method for preserving vaccines, including addingpreservatives e.g., thimerosal. To optimally preserve the activity ofenzymes in the formulation, preferred storage conditions are at −20° C.to −70° C., and can include stabilizers such as glycerol that have lowfreezing points and low vapor pressures. The formulation foradministration may further include buffers and salts, and/or otherwell-known formulation materials, all of which are well-known andrequire little or no experimentation to optimize in a formulation.

The vaccine can also first be created from each venom type in thecombination, and then the separate vaccine types can be mixed to form atoxoid combination, which is then mixed with alum or adjuvants ifdesired, and stored under appropriate conditions to preserve it.

The present invention also includes the process of mapping thegeographical distribution of any venom, toxin, poison, or otherdangerous material generated by plants or animals, and then constructinga polyvalent vaccine against this combination of venoms, toxins,poisons, or other dangerous materials so as to provide improvedprotection for a mammalian subject which is at risk of encountering anyof the toxins, poisons, or other dangerous materials.

These, as well as other materials, components, steps, features, objects,benefits, and advantages, will now become clear from a review of thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a vaccine immunization experiment with mice,where the serum from immunized mice and a control group was reacted withthe venom from the various rattlesnake species and sub-species andregions in California, as shown in the panels. The change in opticaldensity, following reaction of the reacted serum with a secondaryantibody, in vaccinated mice (represented with horizontally hatchedbars) as against control mice (with diagonally hatched bars) is shown inthe panels. An asterisk (*) indicates a statistical difference p<0.001.

FIG. 2 shows the results of a venom challenge experiment, where theimmunized group (dotted lines in each panel) and control group (solidlines in each panel) were injected intraperitoneally with a venom dosagefrom a species or subspecies shown in each panel above LD50, andsurvivorship was monitored for 48 hours. Internal body temperature wasmonitored as a proxy for survivorship and used to determine a humane endpoint for moribund individuals.

DETAILED DESCRIPTION

The method of generating and dosing a mammal with Western Rattlesnaketoxoid vaccine is illustrated further by the following additionalExample 1, and examples of other toxoid vaccines follow. The examplesare not to be construed as limiting the disclosure in any way to thespecific procedures or products described in them, or in any way otherthan as stated in the claims.

The goal in dosing is to expose the subject to all antigens needed tostimulate the animal's immune system to defend against rattlesnakeand/or other snake venom. A number of well-known formulations andadministration protocols can be used to accomplish this, includingintraperitoneal, intravenous, intramuscular, or subcutaneous injection.Any other administration method which can meet the goal stated above canalso be used.

In addition to adjuvants, stabilizers, buffers and salts, theformulation can include any “pharmaceutically acceptable carrier”including, by way of non-limiting example, a pharmaceutically acceptablesolvent, suspending agent, or any other pharmacologically inert vehiclefor delivering toxoids to a subject. Pharmaceutically acceptablecarriers can be liquid, and can be selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties.Examples of pharmaceutically acceptable carriers include, withoutlimitation, water; saline solution; fillers (e.g., lactose and othersugars, gelatin, or calcium sulfate); lubricants (e.g., starch,polyethylene glycol, or sodium acetate); disintegrates (e.g., starch orsodium starch glycolate); and wetting agents (e.g., sodium laurylsulfate).

Example 1. ELISA Protocol of Experiments Demonstrating ProtectionAgainst Several Venom Types with a Polyvalent Vaccine

Vaccine Formulation: one milliliter (mL) of vaccine contains venom fromthe Western Rattlesnake (Crotalus oreganus) in the quantities and wherethe donor snakes were from the regions indicated:

-   -   0.493 mg of venom from donors from the Southern Sierra Nevada        Mountains, California;    -   0.493 mg of venom from donors from the Transverse Mountains,        Calif.;    -   0.014 mg of venom from donors from the San Jacinto Mountains,        Calif.;    -   1:4 Aluminum Hydroxide (Alhydrogel®, InvivoGen); and    -   0.001-0.01% Thimerosal (U.S. Pharmacopeia, 2004).

To determine whether the vaccine could elicit an immune response in amouse model, and show broad protection, an ELISA was conducted andincluded, as control, a comparison of the specific immunity ofvaccinated mice with those immunized using adjuvant alone. Todemonstrate broad protection, specific immunity was determined forindividual mice against venoms from rattlesnakes from eight regions,collectively representing six species and subspecies; that is: twopopulations of Northern Pacific Rattlesnake (Crotalus oreganus oreganus;captured from Napa Valley to Sacramento, Calif.; and New Cuyama toTehachapi, Calif.); two populations of Southern Pacific Rattlesnake(Crotalus oreganus helleri; captured from Southern California from SantaBarbara south, and San Jacinto Mountain Range); Western DiamondbackRattlesnake (Crotalus atrox); Red Diamondback Rattlesnake (Crotalusruber); Southwestern Speckled Rattlesnake (Crotalus mitchelli pyrrhus);and Mojave Rattlesnake (Crotalus scutulatus scutulatus). A group of 20mice were immunized with the vaccine and alum as the adjuvant togenerate polyclonal antibodies using a protocol substantially replicatedfrom Cates et al. (2015), with two vaccine doses there the first dosewas given on day 0 and the second on day 28. Blood serum was collectedfrom the mice on day 56. A different secondary antibody (Santa CruzBiotechnology, m-IgGx BP-HRP, sc-516102) was used instead of theantibody used by Cates et al., which had been discontinued by themanufacturer.

To prepare the plates, 100 micrograms of each of the eight snake venomswas incubated overnight to induce binding to the wells of an individualELISA plate (one plate for each venom type). After washing and blocking,50 μl of mouse serum (diluted 1:8000) was added to the wells. Controlwells for the plate (containing assay reagents) and sample wells, run intriplicate, containing serum from all twenty mice (both those immunizedwith vaccine and adjuvant only control) were run simultaneously againsta single snake venom. Then the assay process was repeated with anothervenom until all eight venom types had been completed. This protocoleliminated plate to plate and run to run variations.

Binding was detected using anti-mouse goat antibodies withHRP-conjugated label, developed with Thermo Scientific Ultra TMB, andthe reaction was stopped with sulfuric acid. Developed plates wereimmediately read at 450 nm. Optical densities measured for each wellwere averaged and a baseline for detection determined by averagingcontrol wells (12×) for each plate. Results are depicted in FIG. 1.

To determine whether there was a significant difference in the specificimmunity of vaccinated and adjuvant-only control mice, Multiple ResponsePermutation Procedures (MRPP) with post hoc Indicator Species Analysiswas performed. This pair of statistical tests is most similar toMultivariate Analysis of Variance (MANOVA) with post hoc tests, and isused to detect statistical differences between known groups and thendetermine what variables are responsible for determining thisdifference. MRPP is a non-parametric multivariate procedure that wasoriginally designed for ecological data that violated assumptions ofconventional (M)ANOVA, especially the requirement fornormally-distributed data. MRPP produces both: (i) a test ofsignificance (p-value) as a criteria for determining whether to rejector fail to reject the null hypothesis that all groups are equal and (ii)a measure of effect size (A-value) known as the chance-correctedwithin-group agreement (McCune and Grace, 2002). The A statisticdescribes within-group homogeneity, where A=1 would indicate that allsamples are identical within groups. McCune and Mefford (2011) haveindicated that A>0.3 is fairly high. Vaccinated mice had significantlyhigher specific immunity compared to mice inoculated with adjuvant only(MRPP: p<0.0001, A=0.48).

Indicator Species Analysis (ISA) was used as a post hoc analysis to MRPPto determine which specific immunities were responsible for drivingseparation between vaccinated and control mice. The procedure calculatesan indicator value (IV) ranging from zero (no indication) to 100(perfect indication).

Any particular ‘species’ (in this case an individual snake venom) withperfect indication would allow determination of whether a mouse was fromthe vaccinated or control group based on the level of specific immunitytowards that venom. In addition to the indicator value calculation, arandomization test was conducted to produce a p value to determinewhether to reject or fail to reject the null hypothesis that theindicator value is not larger than expected by chance (i.e. that a venomis not a good indicator of treatment group compared to random chance).All eight venoms tested had indicator values with p<0.001 and most withstrong indication, IV>80: C. o. oreganus (Napa/Sacramento), IV=80.5; C.o. oreganus (New Cuyama/Tehachapi), IV=81.7; C. o. helleri (So. CA),IV=85.3; C. o. helleri (San Jacinto), IV=71.9; C. atrox, IV=84.5; C.ruber, IV=87.1; C. m. pyrrhus, IV=85.7; C. s. scutulatus, IV=67.5. Thetwo venoms that produced the lowest responses, C. o. helleri (SanJacinto) and C. s. scutulatus, are venoms with an abundance of smallmyotoxins that may represent moieties that are more difficult for miceto develop an immune response against. However, in both cases vaccinatedmice produced detectable quantities of antibodies in a statisticallysignificantly manner; whereas control mice did not have a detectablelevel of antibodies that were able to bind these venoms.

Example 2: Venom Challenge Experiment

Following the same vaccination protocol as in Example 1 experiment,eighty 8-10 week old female CD-1 (Charles Rivers) mice were randomlysorted into two groups and immunized by subcutaneous injection witheither 0.1 mL the vaccine (vaccinated group) or 0.1 mL of AluminumHydroxide (Alhydrogel®, InvivoGen) adjuvant (control group). The firstvaccine dose was given on day 0 and the second on day 28. On day 56,mice were separated into five venom challenge groups and were injectedintraperitoneally with venoms from prevalent rattlesnake subspecies inCalifornia. Challenge doses were set above the Lethal Dose 50% (LD50), astandard value derived for a given toxicant that describes the dose atwhich 50% of a population should succumb to the toxicity of thesubstance. Survivorship was monitored for 48 hours to determine ifvaccination with the vaccine allowed mice to survive a venom challenge(i.e. increased survivorship, decreased mortality). Internal bodytemperature was monitored as a proxy for survivorship and used todetermine a humane end point for moribund individuals. Time to death inminutes was recorded, survivorship curves were plotted, and aKaplan-Meier Survivorship Analysis (Log Rank) was used to determinewhether there were significant differences in survivorship betweenvaccinated and unvaccinated mice. See FIG. 2. In all groups, all control(unvaccinated) mice succumbed to the venom dose, while a significantlygreater number of vaccinated mice survived (p<0.01 for all; at least 50%survivorships for all groups).

Example 3: Vaccine Preparation and Administration

A vaccine created from the venom of Western Rattlesnakes selected fromthree distinct regions (Southern Sierra Nevada Mountains, Calif.;Transverse Mountains, Calif.; San Jacinto Mountains, Calif.) willprovide protection against dangerous components of Western Rattlesnakevenom throughout its range—which includes a number of western states(CA, WA, OR, NV, AZ, UT, ID, WY, CO), and parts of British Columbia andnorthwestern Mexico. Liquid or lyophilized venom samples from WesternRattlesnakes from these three regions are combined in approximatelyequal parts, heated or irradiated to denature the dangerous componentsto produce a toxoid mixture, and mixed with an adjuvant, such as alum,stabilizers, buffers, salts, and one or more pharmaceutically acceptablecarriers, to produce the final formulation of the polyvalent WesternRattlesnake venom vaccine. A preferred final dosage concentration is onemicrogram of the ingredient toxoid per one milliliter dose (i.e., acombination of venom from each of the three California regions abovetotaling one milligram per one milliliter dose) of denatured venom.

To immunize a less than one hundred pound canine or other mammal, onedose is injected, preferably subcutaneously, at least once annually, andpreferably, at least twice with at least a 30 day interval betweendoses, before contact between the mammal and rattlesnakes. Ideally, thedosing schedule would be completed before annual warming following thespring equinox in many of the states that have Western Rattlesnakeand/or other rattlesnake and venomous snake populations. Somespecies/geographically distinct populations of rattlesnakes are activeyear-round in parts of Mexico, California and Arizona so protection ofmammals in these locations may require at least bi-annual dosing asprotection may fail to extend over the entire rattlesnake active period.Further, dogs over 100 pounds or under 25 pounds may benefit from atleast three annual dosings, with the first two doses administered asabove and with a third dose administered 30 days after the second dose.

Example 4. Vaccination Against Viruses and Parasites

Published maps of the distribution of the various mosquito vectors inthe United States can be combined with the distribution of antigens fromseveral mosquito-borne viruses such as West Nile virus, chikungunyavirus, the four common variants of dengue virus, and the Zika virus todetermine which components from these viruses will provide all of thedangerous mosquito-borne parasites (like those causing malaria) andviruses likely to be encountered in the U.S. These virus or parasitesamples are combined and treated to generate toxoids, and produce aformulation for administration, as in Examples 1 and 2. The formulationis administered as in Example 2.

Example 5. Vaccination Against Other Snakes and Venoms

Published maps of the distribution of a variety of snakes and scorpionsnative to the Asia and Africa, including the Palestine Yellow Scorpion(Leiurus quinquestriatus), fat-tailed scorpions such as the blackscorpion (Androctonus crassicauda) and A. amoreuxi, as well as Buthacusarenicola, Buthus occcitanus, and the Egyptian Cobra (Naja haje), theBlack Desert Cobra (Walterinnesia aegyptia), the Puff Adder (Bitisarietans), the Painted Saw-Scaled Viper (Echis coloratus), the IndianSaw-Scaled Viper (E. carinatus), and the Saharan Horned Viper (Cerastescerastes), are utilized in combination with data on the characterizationof each distinct venom component from these species to determine theminimal set of organisms which will provide complete coverage of everydistinct venom component. Liquid venom samples from the identifiedorganisms are combined, treated, formulated, and administered as inExamples 1 and 2.

Any combination of features of the above process and products are withinthe scope of the instant disclosure.

The components, steps, features, objects, benefits, and advantages thathave been discussed are merely illustrative. None of them, nor thediscussions relating to them, are intended to limit the scope ofprotection in any way. Numerous other embodiments are also contemplated.These include embodiments that have fewer, additional, and/or differentcomponents, steps, features, objects, benefits, and/or advantages. Thesealso include embodiments in which the components and/or steps arearranged and/or ordered differently.

Unless otherwise stated, all measurements, values, ratings, positions,magnitudes, sizes, and other specifications that are set forth in thisspecification, including in the exemplary features that follow, areapproximate, not exact. They are intended to have a reasonable rangethat is consistent with the functions to which they relate and with whatis customary in the art to which they pertain.

All articles, patents, patent applications, and other publications thathave been cited in this disclosure are incorporated herein by reference.

Relational terms such as “first” and “second” and the like may be usedsolely to distinguish one entity or action from another, withoutnecessarily requiring or implying any actual relationship or orderbetween them. The terms “comprises,” “comprising,” “including” and anyother variation thereof when used in connection with a list of elementsin the specification or claims are intended to indicate that the list isnot exclusive and that other elements may be included. Similarly, anelement preceded by an “a” or an “an” does not, without furtherconstraints, preclude the existence of additional elements of theidentical type.

The abstract is provided to help the reader quickly ascertain the natureof the technical disclosure. It is submitted with the understanding thatit will not be used to interpret or limit the scope. In addition,various features in the foregoing detailed description are groupedtogether in various embodiments to streamline the disclosure.

TABLE OF REFERENCES

-   McCune, B., & Grace, J. (2002). Analysis of Ecological Communities.    MjM Software, Gleneden Beach, Oreg., U.S.A.-   McCune, B. & Mefford, M. J. (2011). PC-ORD. Multivariate Analysis of    Ecological Data. Version 6.0. MjM Software, Gleneden Beach, Oreg.,    U.S.A.-   Cates, C. C., Valore, E. V., Couto, M. A., Lawson, G. W., &    McCabe, J. G. “Comparison of the protective effect of a commercially    available western diamondback rattlesnake toxoid vaccine for dogs    against envenomation of mice with western diamondback rattlesnake    (Crotalus atox), northern Pacific Rattlesnake (Crotalus oreganus    oreganus), and southern Pacific Rattlesnake (Crotalus oreganus    helleri) venom.” American Journal of Veterinary Research, 76(3),    272-279 (2015).

What is claimed is:
 1. A method of immunizing a mammal againstenvenomation by a rattlesnake comprising: administering an effectiveamount of a polyvalent toxoid vaccine to the mammal, wherein thepolyvalent toxoid vaccine comprises a combination of denatured venomsfrom populations of Western Rattlesnakes selected from the SouthernSierra Nevada Mountains, Calif.; the Transverse Mountains, Calif.; and,the San Jacinto Mountains, Calif.
 2. The method of claim 1 wherein theWestern Rattlesnake is a Southern Pacific, Northern Pacific, Mojave,Southwestern Speckled, Western Diamondback, or Red Diamondbackrattlesnake.
 3. The method of claim 2 wherein the Southern Pacificspecies is the Southern California or San Jacinto subspecies.
 4. Themethod of claim 1 wherein the vaccine is prepared as a solution.
 5. Themethod of claim 4 wherein the solution has a final concentration of 1microgram of toxoid per milliliter of solution.
 6. The method of claim 1further including one or more members selected from the group consistingof: adjuvants, stabilizers, buffers, salts, preservatives and one ormore pharmaceutically acceptable carriers.
 7. The method of claim 6wherein the adjuvant is alum.
 8. The method of claim 6 wherein thepreservative is thimerosal.
 9. The method of claim 1 wherein the venomsare denatured by heating.
 10. The method of claim 1 wherein the venomsare denatured by irradiation.
 11. The method of claim 1 wherein theadministration is by intraperitoneal, intravenous, intramuscular orsubcutaneous injection.
 12. The method of claim 1 wherein the mammal isa dog.
 13. The method of claim 1 wherein the polyvalent toxoid vaccineis administered at a concentration of 1 microgram of toxoid permilliliter of solution.
 14. The method of claim 1 wherein theformulation is administered at least twice annually.
 15. The method ofclaim 14 wherein the formulation is administered at least three timesannually.
 16. The method of claim 14 wherein the second administrationis at least 30 days after the first administration.
 17. The method ofclaim 13 wherein the dog weighs less than 25 or more than 100 pounds.18. The method of claim 1 wherein the polyvalent toxoid vaccine consistsessentially of a combination of denatured venoms from populations ofWestern Rattlesnakes selected from the Southern Sierra Nevada Mountains,Calif.; the Transverse Mountains, Calif.; and, the San JacintoMountains, Calif.
 19. The method of claim 18 wherein the WesternRattlesnake is a Southern Pacific, Northern Pacific, Mojave,Southwestern Speckled, Western Diamondback, or Red Diamondbackrattlesnake.
 20. The method of claim 19 wherein the Southern Pacificsnake is the Southern California or San Jacinto subspecies.